**Preface to "Kinetoplastid Genomics and Beyond"**

Kinetoplastids are a clade of protists located among the earliest-branching eukaryotes. They are mainly recognized because the medical and economic importance of some of their members, e.g. *Trypanosoma* ssp., causing Chagas disease and sleeping sickness in humans, and *Leishmania* spp., causing kala-azar and other types of leishmaniases. Nevertheless, many parasites for plants (*Phytomonas* spp.) and insects (*Leptomonas*, *Crithidia*, and other genera) also belong to this class. In addition, free-living kinetoplastids (bodonids) are abundant and active microbial predators in terrestrial and aquatic ecosystems. Kinetoplastids, together with other two major clades (euglenids and diplonemids), constitute a monophyletic group of flagellates: the Euglenozoa.

Apart from their medical and veterinary relevance, these organisms generate a considerable basic scientific interest due to their bizarre cytology, genome organization, and mechanisms of gene expression regulation. In recent years, the incorporation of "omics"methodologies to the study of these organisms has allowed assembly of the genomes for a growing number of both parasitic and free-living kinetoplastids to analyze changes in gene expression, determine the proteome compendium, establish metabolic pathways, etc.

The aim of this Special Issue was to bring together a set of reviews and research articles on recent and cutting-edge advances in topics related to genome organization, mechanisms of gene expression, and experimental and bioinformatics methodologies, among others. I am grateful to those colleagues who decided contributing to this objective, submitting eleven excellent articles that are now compiled in this book.

In the following paragraphs, with the objective of guiding readers, the contents of the different chapters are briefly summarized, and their connections are highlighted.

As mentioned above, within kinetoplastids, the parasites referred to as trypanosomatids cause severe diseases in humans. Unfortunately, there are no vaccines to prevent these infections, and the available drugs to control these diseases are far from ideal due to host toxicity, limited access, and increasing rates of drug resistance. Here, in the first chapter, Bhattacharya and colleagues present a comprehensive review on current chemotherapy against tripanosomatids and, more importantly, describe the technological advances in parasitology, chemistry, and genomics that have brought improved compound screening technologies and incorporated novel drug concepts. As documented in this review, these new approaches are uncovering new lead compounds and, consequently, more effective treatments are envisioned for the near future [1].

The need for new drugs for treatment and the problem of drug resistance is also illustrated in the article by Ghosh et al [2]. Artemisinin, a drug used for malaria treatment, is being explored as a candidate drug for combating leishmaniasis. Nevertheless, apart from its efficacy, it is mandatory to establish the easiness by which parasites might create resistance. In this study, by comparative genomics and transcriptomics analyses of in vitro-adapted artesunate-resistant Leishmania donovani parasites, the authors have outlined the molecular basis underlying artemisinin resistance in *Leishmania* parasites.

Trypanosomatids exhibit a number of highly peculiar molecular features. Among them, a remarkable peculiarity is the genome structure: genes are organized into large collinear clusters, but contrary to prokaryotic polycistronic units, the genes present have no common nor akin function. Other modulators of genome structure are retroposons and gene families comprised of abundant and sequence variable members. These genomic peculiarities are illustrated in the chapter by Herreros-Cabello et al. [3], who reviewed current knowledge on *Trypanosoma cruzi* genome architecture and plasticity. Within the study of the *T. cruzi* genome, the chapter by Bernardo et al [4] shows an in-depth analysis of the Retrotransposon Hot Spot (RHS) gene family. The RHS family is the largest gene family existing in the *T. cruzi* genome, but, at the same time, the most enigmatic regarding their cellular functions. Based on their nuclear location, the authors suggest that RHS proteins might be involved in the control of the chromatin structure and gene expression along the parasite life cycle. Apart from multigenic families, the *T. cruzi* genome is populated by interspersed repetitive DNA elements that amount for a significant fraction of its genomic content. As suggested by Calderano et al. [5], these repeated elements, often located at the 3'-untranslated regions (3'-UTRs) of genes, may be essential players for the mechanisms regulating gene expression. The kinetoplastids have a unique reliance on post-transcriptional control of gene expression, and the uncovering of cisand trans-acting regulators is a basic step towards the understanding of how these organisms regulate mRNA and proteins levels.

Crucial components of genome architecture are origins of replication (ORIs), i.e., the places in which DNA replication initiates. The activation (firing) of ORIs is an extremely regulated process, as the cell viability depends on the complete replication of every chromosome within a precise phase (S-phase) of the cell cycle. In kinetoplastids, our knowledge about the number of ORIs required to replicate their genomes is limited. In the chapter by da Silva et al. [6], a bioinformatic tool designed to calculate the minimum number of ORIs required to duplicate an entire chromosome within the S-phase duration in trypanosomatids (*T. cruzi*, *Leishmania major*, and *T. brucei*) and yeasts (*Saccharomyces cerevisiae* and *Schizosaccharomyces pombe*) is described.

Complete and well-annotated genomes represent the ultimate resource for genome-wide scale studies, such as transcriptomic and proteomic analyses. However, as documented in the chapter by Sanchiz et al. [7], a proteogenomic approach should be considered as a first choice when determining the experimental proteome for a given organism, *Leishmania infantum* in this instance. This strategy would allow the uncovering of new protein-coding genes and, consequently, to improve gene annotations.

As mentioned above, kinetoplastids depend on post-transcriptional mechanisms for gene expression regulation. This includes post-transcriptional protein modifications, which contribute to cellular phenotypes by altering protein abundance, function, and localization. In the chapter by Bea et al. [8], it is documented that the machinery of modification of polypeptides by the covalent attachment of small ubiquitin-like modifier (SUMO) moiety (SUMOylation of proteins) is essential for *Leishmania donovani* viability and infectivity. In this study, the CRISPR-Cas9-mediated gene edition system was used. The CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)–Cas9 (CRISPR-associated protein 9) methodology is revolutionizing in vivo studies aimed to decipher gene function in many organisms. The chapter by Adaui et al. [9] illustrated the use of this technique in *Leishmania braziliensis*. The authors successfully applied a cloning-free, PCR-based CRISPR–Cas9 technology to inactivation of the two alleles of two well-characterized heat-shock genes, HSP23 and HSP100. The detailed description of the technique and the compendium of methods used in the study for the characterization of the mutant lines make this chapter a methodological reference article.

The evolutionary and biogeographical history of kinetoplastids is fascinating, even though it remains a hotly debated topic, and multiple hypotheses have been proposed. The chapter by Cantanhede et al. [10] reviewed our current knowledge on the origin of ˆ *Leishmania* parasites, adding a new player, the RNA viruses identified in many species of this genus. Phylogenetic analyses of the endosymbiotic Leishmania viruses and the *Leishmania* species harbouring them suggest a long coevolutionary relationship, which would enhance parasite survival and virus fitness during leishmaniasis.

As stated above, kinetoplastids belong to the Euglenozoa group, an evolutionary ancient phylum of flagellate eukaryotes. In the final chapter of this book, Cordoba and co-workers [11] present a study aimed to generate a comprehensive transcriptome for *Euglena gracilis*, a known photosynthetic microeukaryote considered as the product of a secondary endosymbiosis between a green alga and a phagotrophic unicellular protist, an evolutionary relative of kinetoplastids. Thus, analysing *E. gracilis* genomic and transcriptomic information is a way to approach the evolution of parasitism. In this regard, the authors of this study show evidence that trans-splicing mechanisms (typical of trypanosomatids) are also occurring in a large percentage of the *E. gracilis* transcripts.

In summary, this collection of eight original research articles and three reviews covers a wide range of topics in the field of kinetoplastids. In addition, readers can find a compendium of experimental methods and bioinformatics tools.

Finally, I would like to express my gratitude to the contributing authors and thanks to Maggie Miao for her invaluable editorial assistance. This book is also dedicated to my daughter Carmen.

References

1. Bhattacharya, A.; Corbeil, A.; Do Monte-Neto, R.L.; Fernandez-Prada, C. Of drugs and trypanosomatids: New tools and knowledge to reduce bottlenecks in drug discovery. Genes (Basel). 2020, 11, 1–24.

2. Ghosh, S.; Verma, A.; Kumar, V.; Pradhan, D.; Selvapandiyan, A.; Salotra, P.; Singh, R. Genomic and transcriptomic analysis for identification of genes and interlinked pathways mediating artemisinin resistance in leishmania donovani. Genes (Basel). 2020, 11, 1362, doi:10.3390/genes11111362.

3. Herreros-Cabello, A.; Callejas-Hernandez, F.; Giron ´ es, N.; Fresno, M. Trypanosoma cruzi ` genome: Organization, multi-gene families, transcription, and biological implications. Genes (Basel). 2020, 11, 1196.

4. Bernardo, W.P.; Souza, R.T.; Costa-Martins, A.G.; Ferreira, E.R.; Mortara, R.A.; Teixeira, M.M.G.; Ramirez, J.L.; Da Silveira, J.F. Genomic organization and generation of genetic variability in the RHS (Retrotransposon hot spot) protein multigene family in Trypanosoma cruzi. Genes (Basel). 2020, 11, 1–19, doi:10.3390/genes11091085.

5. Calderano, S.G.; Nishiyama Junior, M.Y.; Marini, M.; Nunes, N. de O.; Reis, M. da S.; Patane,´ J.S.L.; da Silveira, J.F.; da Cunha, J.P.C.; Elias, M.C. Identification of novel interspersed DNA repetitive elements in the trypanosoma cruzi genome associated with the 3UTRs of surface multigenic families. Genes (Basel). 2020, 11, 1235, doi:10.3390/genes11101235.

6. da Silva, M.S.; Vitarelli, M.O.; Souza, B.F.; Elias, M.C. Comparative analysis of the minimum number of replication origins in trypanosomatids and yeasts. Genes (Basel). 2020, 11, 523, doi:10.3390/genes11050523.

7. Sanchiz, A.; Morato, E.; Rastrojo, A.; Camacho, E.; Gonz ´ alez-de la Fuente, S.; ´ Marina, A.; Aguado, B.; Requena, J.M. The Experimental Proteome of Leishmania infantum Promastigote and Its Usefulness for Improving Gene Annotations. Genes (Basel). 2020, 11, E1036, doi:10.3390/genes11091036.

8. Bea, A.; Krober-Boncardo, C.; Sandhu, M.; Brinker, C.; Clos, J. The leishmania donovani ¨ SENP protease is required for SUMO processing but not for viability. Genes (Basel). 2020, 11, 1–16, doi:10.3390/genes11101198.

9. Adaui, V.; Krober-Boncardo, C.; Brinker, C.; Zirpel, H.; Sellau, J.; Ar ¨ evalo, J.; Dujardin, J.C.; ´ Clos, J. Application of crispr/cas9-based reverse genetics in leishmania braziliensis: Conserved roles for hsp100 and hsp23. Genes (Basel). 2020, 11, 1–24, doi:10.3390/genes11101159.

10. Cantanhede, L.M.; Mata-Somarribas, C.; Chourabi, K.; Pereira da Silva, G.; Dias Das Chagas, ˆ B.; de Oliveira R. Pereira, L.; Cortes Boit ˆ e, M.; Cupolillo, E. The maze pathway of coevolution: A ´ critical review over the leishmania and its endosymbiotic history. Genes (Basel). 2021, 12, 657.

11. Cordoba, J.; Perez, E.; Van Vlierberghe, M.; Bertrand, A.R.; Lupo, V.; Cardol, P.; Baurain, D. De Novo Transcriptome Meta-Assembly of the Mixotrophic Freshwater Microalga Euglena gracilis. Genes (Basel). 2021, 12, 842, doi:10.3390/genes12060842.

> **Jose M. Requena** *Editor*

*Review*
